Chemicals can cause cancer through a variety of mechanisms. Genotoxic carcinogenic compounds may lead to damage of the genetic material, either directly by covalently binding to DNA or indirectly by interfering with the mitotic machinery, ultimately leading to mutations in genes or large aberrations in chromosomes (Hayashi, Y. (1992) Overview of genotoxic carcinogens and non-genotoxic carcinogens Exp Toxicol Pathol 44, 465-71). Genotoxic carcinogenic compounds are also often called genotoxic carcinogens and their mode of action is also referred to as genotoxicity.
If tumour suppressor genes are inactivated or proto-oncogenes are activated by this process, this may lead to uncontrolled growth of cells, in particular to cancer formation. The mode of action of non-genotoxic carcinogens can be very diverse, but does not include damage to DNA or chromosomes, and may involve stimulation of cell proliferation, suppression of apoptosis, induction of oxidative stress, activation of biotransformation enzymes, suppression of the immune system, etc (Shaw, I. C. & Jones, H. B. (1994) Mechanisms of non-genotoxic carcinogenesis Trends Pharmacol Sci 15, 89-932).
Polycyclic aromatic hydrocarbons (PAH) comprise of a large group of structurally related compounds, of which many are carcinogens. Humans are daily exposed to PAH as they are widely present in the environment and in food. Many carcinogenic PAH, such as benzo[a]pyrene (BaP), share both genotoxic and non-genotoxic properties.
During several enzymatic steps involving cytochrome P450 enzymes and epoxide hydroxylase, BaP is converted into the ultimate genotoxic carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE can react with nucleophylic groups in DNA, thereby forming DNA adducts, mainly consisting of BPDE-N2deoxyguanosin (BPDE-dG) (Cheng, S. C., Hilton, B. D., Roman, J. M. & Dipple, A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide Chem Res Toxicol 2, 334-40). If not removed by DNA repair systems, these adducts lead to mutations during replication (Moriya, M., Spiegel, S., Fernandes, A., Amin, S., Liu, T., Geacintov, N. & Grollman, A. P. (1996) Fidelity of translesional synthesis past benzo[a]pyrene diol epoxide-2′-deoxyguanosine DNA adducts: marked effects of host cell, sequence context, and chirality Biochemistry 35, 16646-51). BaP can also be transformed into radical cations by CYP peroxidase (Cavalieri, E. L. & Rogan, E. G. (1995) Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons Xenobiotica 25, 677-88), and into reactive and redox-active o-quinones involving aldo-kto reductases, which also can form stable and depurinating DNA adducts (Penning, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D., Palackal, N. T. & Tsuruda, L. S. (1999) Dihydrodiol dehydrogenases and polycyclic aromatic hydrocarbon activation: generation of reactive and redox active o-quinones Chem Res Toxicol 12, 1-18 and Balu, N., Padgett, W. T., Lambert, G. R., Swank, A. E., Richard, A. M. & Nesnow, S. (2004) Identification and characterization of novel stable deoxyguanosine and deoxyadenosine adducts of benzo[a]pyrene-7,8-quinone from reactions at physiological pH Chem Res Toxicol 17, 827-38). Auto-oxidation of the intermediate catechols and/or subsequent redox cycling of the o-quinones generates reactive oxygen species (ROS) capable of modifying DNA, leading to the formation of oxidised nucleotides like 8-hydroxy-deoxyguanosine (8-OHdG) (Caino, M. C., Oliva, J. L., Jiang, H., Penning, T. M. & Kazanietz, M. G. (2007) Benzo[a]pyrene-7,8-dihydrodiol promotes checkpoint activation and G2/M arrest in human bronchoalveolar carcinoma H358 cells Mol Pharmacol 71, 744-50). These processes all comprise the genotoxic properties of BaP.
Cisplatin is another genotoxic carcinogenic agent that is often used as a chemotherapeutic agent in the treatment of cancer. Cisplatin damages DNA by forming DNA-protein cross-links. See, Costa, M., et al. (1997) J. Toxicol. Environ. Health 50(5), 433-449; and Zhitkovich, A. and Costa, M. (1992) Carcinogenesis 13(8), 1485-1489.
Genetic toxicology testing in the pharmaceutical industry provides an assessment of genotoxic risk associated with the use of drugs. Since the beginning of genotoxicity testing in the early 1970s, many different test systems have been developed and used. No single conventional test is capable of detecting all genotoxic agents. Therefore, genotoxic evaluation of pharmaceutical compounds involves the use of a standard battery of in vitro and in vivo assays. See ICH (1997) Harmonized tripartite guideline, genotoxicity: a standard battery for genotoxicity testing of pharmaceuticals. Recommendations for adoption at step 4 of the ICH process on July 1997 by the ICH Steering Committee (final draft). These tests include bacterial reverse-mutation tests, in vitro tests for chromosomal damage (e.g., cytogenetic assays and in vitro mouse lymphoma thymidine kinase assay) and in vivo tests for chromosomal damage (e.g., rodent micronucleus test).
Molecular biology and recombinant technology provide additional methods by which genotoxicity may be measured. For example, differential gene expression technology may be used to study changes in gene expression of cells exposed to drug and chemical substances. Differential gene expression may be measured using various techniques known to those with skill in the art, including, gel electrophoresis and polynucleotide microarrays.
Genotoxic stress triggers a variety of biological responses including the transcriptional activation of genes regulating DNA repair, cell survival and cell death. Factors that may cause genotoxic stress include exposure to chemical and biological agents and radiation.
GADD45 has been identified as a gene that is induced by DNA damaging factors such as ultraviolet and gamma radiation, the alkylating agent, methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorine and hydrogen peroxide. See; Fornace, A. J., et al. (1988) Proc. Natl. Acad. Sci. USA 85, 8800-8804; Papathanasiou, M. A., et al. (1991) C. Mol. Cell. Biol. 11, 1009-1016; and Takekawa, M. and Saito H. (1998) Cell 95(4), 521-530.
US2007224609 relates to methods for detecting for the presence of an agent that putatively causes or potentiates DNA damage comprising subjecting a cell to an agent; and monitoring the expression of the reporter protein from the cell. The cells as used therein contain a DNA sequence encoding a reporter protein operatively linked to a human GADD45alpha gene promoter and a human GADD45alpha gene regulatory element arranged to activate expression of the DNA sequence in response to DNA damage. The invention also concerns expression cassettes, vectors and cells which may be used according to such a method and also modified media that may be employed in fluorescence assays and in preferred embodiments of the method of the invention.
Aubrecht, J., et al. (1999) Toxicol. Appl. Pharmacol. 154(3), 228-235 discloses that metallocene molecules containing vanadium, although structurally similar to cisplatin and causing cytotoxicity via apoptosis as does cisplatin, do not cause DNA double strand breaks measured by the yeast DEL recombination assay and do not activate GADD45 promoter activity.
WO 97/13877 and related U.S. Pat. No. 6,228,589 disclose methods for assessing the toxicity of a compound in a test organism by measuring gene expression profiles of selected tissues.
U.S. Pat. No. 5,811,231 describes methods and diagnostic kits for identifying and characterizing toxic compounds, wherein the methods and kits measure transcription or translation levels from genes linked to native eukaryotic stress promoters.
Rockett, John C., et al. (2000) Xenobiotica 30, 155-177 describes DNA arrays and methods of preparing and using DNA arrays and various applications for which DNA arrays may be used, including toxicological applications.
US2008138820 describes a method for determining the effect of a test agent on a phenotypic property of a specific cell expressing a number of reporter genes responsive to the test agent.
WO2006056340 describes a method for determining the genotoxicity of a test compound, said method comprising the steps of contacting a viable test cell with said test compound and determining the change in expression level of an indicator gene selected from the group consisting of Prelp (Proline arginine-rich end leucine-rich repeat); Sesn2 (Sestrin 2); 4833427G06 Rik (RIKEN cDNA); Dda3 (Differential display and activated by p53); Usp30 (Ubiquitin specific protease 30); 0610013D04 Rik (RIKEN cDNA); Slcl9a2 (Solute carrier family 19 (thiamine transporter, member 2); Trp53inpl (Transformation related protein 53, inducible nuclear protein 1); D4Ertd421e (DNA segment, Chr 4, ERATO Doi 421, expressed); Shcbpl (She SH2-domain binding protein 1); MM67 (Antigen identified by MAb Ki67); Phex (Phosphate regulating neutral endo-peptidase (X chromosome)); Tkl (Thymidine kinase 1); Mmhead (Mus musculus 15 day embryo head cDNA clone); Osbpl6 (Oxysterol binding protein-like 6); Mphosphl (M-phase phosphoprotein); Ephxl (Epoxide hydrolase 1 (microsomal xenobiotic hydrolase)); Top2a (Topoisomerase (DNA) II alpha); Ccngl (Cyclin GI); Plf (Proliferin); Np95 (Nuclear protein 95); Rad51apl (RAD51-associated protein 1); Nos3 (Nitric oxide synthase 3, endothelial cell); 2610005B21 Rik (RIKEN cDNA); Brcal (Breast cancer 1); Stk18 (Serine/threonine kinase 18); Calmbpl (Calmodulin binding protein 1); Lekl (Leucine, glutamic acid, lysine family 1 protein); Smc2U (SMC2 structural maintenance of chromosomes 2-like 1); E2f7 (E2F transcription factor 7); Hmmr (Hyaluronan mediated motility receptor (RHAMM)); Nusapl (Nucleolar and spindle associated protein 1); Fbxo5 (f-box only protein 31); Slcl9a2 (Solute carrier family 19 (thiamine transporter), member 2); 9030617003 Rik (RIKEN cDNA); Ly6e (Lymphocyte antigen 6 complex, locus E); 6530401 L14 Rik (RIKEN cDNA); Mad3 (Max dimerization protein 3); Hmgb2 (High mobility group box 2); Kifll (Kinesin 11); Mad2ll (MAD2 (mitotic arrest deficient, homolog)-like 1 (yeast)); Asflb (ASFI anti-silencing function 1 homolog B (Saccharomyces)); Mcm3 (Minichromosome maintenance deficient 3 (Saccharomyces)); MGC: 32192 (Mus musculus cDNA clone MGG32192 IMAGE:5006129); Foxml (Forkhead box MI); Anxa8 (Annexin A8); Slc35a5 (Solute carrier family 35, member A5); E030024M05 Rik (RIKEN cDNA); Cks2 (CDC28 protein kinase regulatory subunit 2); Cilp (Cartilage intermediate layer pro); Tacc3 (Transforming, acidic coiled-coil containing protein 3); Prcl (Protein regulator of cytokinesis 1); 2610509G12 Rik (RIKEN cDNA); 2810417H13 Rik (RIKEN cDNA); Pbk (PDZ binding kinase); Capn[beta] (Calpain 6); Gmnn (Geminin); Mcmd4 (Minichromosome maintenance deficient 4 homolog); Ccna2 (Cyclin A2); Polal (DNA polymerase alpha 1, 180 kDa); Hmgb3 (High mobility group box 3); Tagln (Transgelin (smooth muscle 22 protein)); 1600013K19 Rik (RIKEN cDNA); Serpinel (Ser (or Cys) proteinase inhibitor, clade E, member 1); Wigl (Wild-type p53-induced gene 1); Hgf (Hepatocyte growth factor (scatter factor)); Gnpi (Glucosamine-6-phosphate deaminase); Birc5 (Baculoviral IAP repeat-containing 5); Priml (DNA primase, p49 subunit); Rbll (Retinoblastoma-like 1 (plO7)); Pcna (Proliferating cell nuclear antigen); E130315B21 Rik (RIKEN cDNA); 2610019103 RIK (RIKEN cDNA); wherein an increase in expression of at least 1.5-fold indicates that said test compound exhibits genotoxicity.
WO2007073774 concerns the use of P130Cas/BCAR1 protein and/or encoding gene for the screening for substances useful in the treatment of tumors. It also concerns a method for the diagnosis of malignant cell growth comprising the measuring the expression of P130Cas/BCAR1 gene. It also concerns non-human transgenic animals as model study for human pathologies, preferably cancer, being transgenic for having altered P130Cas/BCAR1 and Neu-T expression.
WO2005081783 provides a method of detecting exposure to a genotoxic agent in a live subject, comprising the steps of collecting a sample from said subject and detecting the presence of FANCD2-containing foci in said sample, wherein the presence of foci is indicative of exposure to a genotoxic agent.
Patent application UA14610U describes a method for detecting genotoxic and cytotoxic action of antihelminthics which comprises the steps of treating a biological test object with a preparation and a subsequent analysis of samples taken from the test object. Cells of mouse marrow and pig blood lymphocytes are used as the test objects. DNA is extracted from the cell suspensions and analysed for damages using gel electrophoresis.
US2004197855 discloses a process for the determination of genotoxicities characterised in that cells comprising a set of fluorescent or luminescent selection markers are brought into contact with a test compound, wherein the appearance, increase, or decrease in fluorescent or luminescent cells corresponding to the activity of the wild-type gene product of the selection markers indicates the genotoxic effect of the test compound.
US2003203388 deals with a method of characterizing an agent, comprising the steps of treating a mammal cell or a mammal with an agent; and characterizing the DNA-interacting genotoxicity of said agent by determining the effect of said agent on expression in said mammal cell or mammal of at least one gene selected from the group consisting of genes with accession numbers X54149, 000937, AV138783, A1847051, AF055638, A1461837.
U.S. Pat. No. 6,713,261 discloses a method for assessing in vitro the genotoxicity of a compound, which consist in contacting said compound with at least a cell or cell type overexpressing bcl2 protooncogene and/or related anti-apoptotic protein, and observing the genotoxic effects of said compound on said cell.
In spite of all the above described methods, there exists a need to further identify, characterize and understand the mechanism of action of toxicologically relevant genes in order to simplify the development, screening, and testing of new drug and chemical substances.